We investigated the pathophysiologic mechanism of familial/sporadic generalized glucocorticoid resistance syndrome, a prototype of glucocorticoid resistance caused by mutations in the glucocorticoid receptor (GR) gene. We found three new heterozygotic cases with mutations in the GR gene (GR V423A, GR V575G and GR H726R) in collaboration with Dr. Evangelia Charmandari, the Univ of Athens Medical School, Athens, Greece. We are currently analyzing their molecular defects. We have almost completed the computer-based 3-dimensional structural analysis for the ligand-binding domain of all pathologic GR mutations ever reported in addition to these newly identified mutations by using the algorism we established for GR R714Q in the last year. We also encountered a 7-year boy with mild resistance to glucocorticoids, androgens and thyroid hormones. Array-based comparative genomic hybridization analysis showed that this patient has 1.1 Mb size heterozygotic 16p11.2 microdeletion, while the siRNA-based screening and subsequent molecular analyses revealed that heterozygotic deletion of the ZNF764 gene by his microdeletion is responsive for his multi-hormone resistance, as this protein acts as a common coactivator for the glucocorticoid, androgen and thyroid hormone receptors. This is the first case demonstrating resistance to multiple steroid hormones with identification of the causative gene. We are now preparing a manuscript based on these results. The liver X receptors (LXRs), which belong to the nuclear receptor superfamily, mediate the biologic actions of various lipids, such as the cholesterol metabolites oxysterols, prostanoids and some fatty acids, by directly binding to these molecules. LXRs exist as two subtypes, LXR&#945;and LXR&#946;, which display distinct patterns of tissue expression. Once LXRs bind their lipid ligands, they form a heterodimer with the retinoid X receptor (RXR), and stimulate the transcription of an array of genes involved in the absorption, efflux, transport, and excretion of cholesterol and other lipids. LXRs also regulate glucose metabolism by decreasing the expression of its rate-limiting enzymes G6Pase and PEPCK, and have anti-inflammatory activity by repressing a set of inflammatory genes in macrophages and other immune cells. Overexpression/ligand (GW3965) activation of LXRs/RXRs repressed GR-stimulated transactivation of glucocorticoid response element (GRE)-driven promoters in a gene-specific fashion, and activation of LXRs by GW3965 attenuated dexamethasone-stimulated elevation of circulating glucose in rats and suppressed dexamethasone-induced mRNA expression of hepatic glucose-6-phosphatase (G6Pase) in rats, mice and human hepatoma HepG2 cells. Mechanistically, we found that LXR&#945;/RXR&#945;bound GREs and inhibited GR binding to these DNA sequences in a gene-specific fashion. We propose that administration of LXR agonists may be beneficial in glucocorticoid treatment- or stress-associated dysmetabolic states by directly attenuating the transcriptional activity of the GR on glucose and/or lipid metabolism. Circulating levels of glucocorticoids fluctuate naturally in a circadian fashion, and regulate the transcriptional activity of the GR in target tissues. The basic helix-loop-helix protein CLOCK, a histone acetyltransferase (HAT), and its heterodimer partner BMAL1 are self-oscillating transcription factors that generate circadian rhythms both in the central nervous system and periphery. We previously reported that CLOCK/BMAL1 repressed GR-induced transcriptional activity by acetylating GR at several lysine residues located in its hinge region and by suppressing binding of GR to promoter GREs. These findings indicate that CLOCK/BMAL1 functions as a reverse phase negative regulator of glucocorticoid action in target tissues, possibly by antagonizing the biologic actions of diurnally fluctuating circulating glucocorticoids. We thus performed a human study in which we sampled peripheral blood in the morning and evening from normal subjects, and measured mRNA expression of known glucocorticoid-responsive genes and GR acetylation in circulating lymphocytes. We found that GR was acetylated higher in the morning than in the evening, positively correlating with mRNA expression of CLOCK and BMAL1, while circulating glucocorticoid-stimulated mRNA expression of glucocorticoid responsive genes were repressed by CLOCK/BMAL1 in a gene-specific fashion. These results indicate that the peripheral CLOCK system negatively regulates GR transcriptional activity through acetylation of GR not only in cultured cells but also in humans. In another clinically oriented study in which we measured mRNA expression of 190 GR action-regulating and glucocorticoid-responsive genes in subcutaneous fat biopsied from 25 obese subjects, we found that the morning/evening cortisol ratio, an indicator for the amplitude of circadian rhythmicity of circulating cortisol, negatively correlated with mRNA expression of the nicotinamide phosphoribosyltransferase (NAMPT), which is a rate-limiting enzyme for the production of intracellular NAD+. It is known that NAMPT/NAD+ is necessary for maintaining circadian rhythms of the CLOCK transcriptional system. Our results thus indicate that diurnal fluctuation of circulating glucocorticoids provides negative input to local CLOCK activity. In connection with our study on circadian rhythm, we found one interesting family with seasonal alteration of circadian rhythmicity in collaboration with Dr. F. Halberg, the Univ. of Minnesota. The proband, 61-year old female, has suffered from an annual cycle of severe fatigue including the inability to get out of bed, which lasts 2-3 months in the summer and the winter. She, however, is free from symptoms with high achievement during the unaffected months between these bad periods. The proposita demonstrated elongation of circadian rhythmicity (24.84 hours) in blood pressure and vigor rates during the affected periods, while she was in normal 24-hour rhythm in the unaffected months. The cycle of 24.84 hours is exactly the double of the tidal cycle, which is hypothesized to come about gravity-changes caused by movement of the moon around the earth. To identify genetic cause(s) of these manifestations, we have started whole exome analysis, sequencing the entire coding region of genome in 3 affected subjects and 5 unaffected members of the family.